Among glycated hemoglobins in which sugar is bound to hemoglobin (hereinafter, referred to as “Hb”) in blood, hemoglobin A1c (hereinafter, referred to as “HbA1c”) in which the N-terminal valine residue of the hemoglobin β-chain is glycated with glucose, clinically reflects the average blood glucose level of the past one to two months. Therefore, HbA1c is widely used as a marker appropriate for the diagnosis of diabetes or a progress observation of diabetes.
As the method for measuring HbA1c, HPLC methods and immunological assay methods have been used, but because the measurement objects subtly varies with the type of the HPLC column or among the reagent manufacturers, the standardization of the assay is requested. From such a viewpoint, the working group for HbA1c standardization of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) determined a standard assay method (hereinafter, referred to as “IFCC reference method”) (Non-Patent Document 1). The method involves digesting a red blood cell lysate with a protease, thereby extracting the N-terminal hexapeptide of the hemoglobin β-chain (VHLTPE) (SEQ ID NO: 1), determining the amounts of the glycated N-terminal hexapeptide (f-VHLTPE) (SEQ ID NO: 11) of the HbA1c β-chain and the unmodified N-terminal hexapeptide (VHLTPE) (SEQ ID NO: 1) of the β-chain of hemoglobin AO (hereinafter, referred to as “HbA0”) in the extract, and calculating the HbA1c content (%) by the formula shown below:HbA1c content (%)=(Amount of f-VHLTPE (SEQ ID NO: 11)/(amount of f-VHLTPE (SEQ ID NO: 11)+amount of VHLTPE (SEQ ID NO: 1)))×100=(Amount of HbA1c/(amount of HbA1c+amount of HbA0))×100
However, since the method for measuring the glycated hexapeptide content according to this method makes use of HPLC-MS or HPLC-capillary electrophoresis, the method requires expensive apparatuses, and the measurement can be made only in particular facilities. Furthermore, there is a problem that the measurement values obtained by this method are different greatly from the HbA1c values obtained by conventional methods (Non-Patent Document 2).